High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation.

Cell Reports
M. LegutNeville E Sanjana

Abstract

A key limitation of the widely used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG), but their activity has been measured at only a small number of endogenous sites. Here, we devise a high-throughput Cas9 pooled competition screen to compare the performance of Cas9 variants at thousands of genomic loci for gene knockout, transcriptional activation, and inhibition. We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cleavage. Of the PAM-flexible variants, we find that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combine xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.

References

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Citations

Oct 2, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Jasper Edgar NeggersDirk Daelemans
Oct 17, 2020·The Journal of Biological Chemistry·David L ProleNick S Jones
Feb 5, 2021·Journal of Clinical Medicine·Christi T Salisbury-Ruf, Andre Larochelle
Jan 18, 2021·The Journal of Biological Chemistry·David L ProleNick S Jones
Apr 7, 2021·Nucleic Acids Research·Maria Claudia Villegas KcamJames Chappell
Nov 7, 2020·Cell Stem Cell·Delilah HendriksBenedetta Artegiani
May 18, 2020·Nature Communications·Gita Naseri, Mattheos A G Koffas
Jun 15, 2021·EcoSal Plus·Nicholas Backes, Gregory J Phillips
Jun 17, 2021·Nucleic Acids Research·Weiwei ZhangJiazhi Hu
Jul 31, 2021·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Yuanyuan Li, Li-Quan Zhou
Oct 19, 2021·Frontiers in Medicine·Jennifer Hernández-JuárezShyamanga Borooah

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Datasets Mentioned

BETA
GSE143892

Methods Mentioned

BETA
gene knockout
Illumina sequencing
fluorescence-activated cell sorting
FACS
flow cytometry
transfection
PCR

Software Mentioned

GraphPad Prism
xCas9
CRISPResso2
ImageJ
SunTag
PyMOL
LOESS
Gviz
benchling
NG

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